The cell types in our bodies fall into two basic classes, termed epithelial cells and mesenchymal cells. Epithelial cells are essentially stationary but mesenchymal cells are potentially able to move. Although these classes are fundamentally very different, epithelial to mesenchymal transition (EMT) occurs when the body needs to move functionally specialized cells from one place to another. This is a carefully defined and highly regulated process in development, but deregulation of the EMT during neoplasia is emerging as a major mechanism whereby epithelial cancers (carcinomas) metastasise to other parts of the body, which is the major cause of death in breast cancer. Expression of the mesenchymal cell intermediate filament protein vimentin (VIM) has been used traditionally as a marker of the EMT. Additional "early" EMT markers are needed, since VIM can be expressed by epithelial cells prior to the EMT, while EMT-derived VIM + cells may have already fled the tumor. Our recent studies have shown that EGF can cause EMT-like changes to occur in the VIM+ PMC42-ET subline, and can partially stimulate PMC42 parental cells. This stepwise progression from epithelial to mesenchymal status in the same cell line provides a model system for elucidation of new "early" markers of the EMT and new potential targets for breast cancer therapy. High throughput cDNA array analysis of gene expression profiles in the PMC42 system with the proven Incyte/Synteni system will identify candidate gene expression changes which indicate different stages of the EMT in breast cancer cells. Integration of our data with profiles from the NC160 cell line analysis will provide a context for the PMC42 profiles, and refinement of the breast cancer data in that set. Important changes will be validated with real time quantitative RT-PCR analysis of laser capture micro-dissected breast cancers of differing VIM status, and candidates showing promise as early markers will be tested for predictive power in a well defined cohort of over 367 subjects for which archival material and 5 year follow-up is available. The functional importance of selected candidate genes will be tested by transfection into PMC42 cells, followed by studies of cellular morphogenesis and in vitro invasiveness. Further studies of in vivo metastasis will be the subject of subsequent applications. Our goal is to obtain leads for new diagnostic markers and new therapeutic targets related to the EMT in human breast cancer.